Background Transfusion-related sepsis remains an important hospital infection control challenge. Investigation of septic transfusion events is often restricted by the limitations of bacterial culture in terms of time requirements and low yield in the setting of prior antibiotic administration. Methods In 3 gram-negative septic transfusion cases, we performed metagenomic next-generation sequencing (mNGS) of direct clinical blood specimens in addition to standard culture-based approaches utilized for infection control investigations. Pathogen detection leveraged IDSeq, a new open-access microbial bioinformatics portal. Phylogenetic analysis was performed to assess microbial genetic relatedness and understand transmission events. Results mNGS of direct clinical blood specimens afforded precision detection of pathogens responsible for each case of transfusion-related sepsis and enabled discovery of a novel Acinetobacter species in a platelet product that had become contaminated despite photochemical pathogen reduction. In each case, longitudinal assessment of pathogen burden elucidated the temporal sequence of events associated with each transfusion-transmitted infection. We found that informative data could be obtained from culture-independent mNGS of residual platelet products and leftover blood specimens that were either unsuitable or unavailable for culture or that failed to grow due to prior antibiotic administration. We additionally developed methods to enhance accuracy for detecting transfusion-associated pathogens that share taxonomic similarity to contaminants commonly found in mNGS library preparations. Conclusions Culture-independent mNGS of blood products afforded rapid and precise assessment of pathogen identity, abundance, and genetic relatedness. Together, these challenging cases demonstrated the potential for metagenomics to advance existing methods for investigating transfusion-transmitted infections.

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42Main Point: Transfusion-transmitted infections cause significant morbidity and are challenging 43 to prevent and diagnose. We found that culture-independent metagenomic sequencing of blood 44 products afforded rapid and precise assessment of pathogen identity, abundance and genetic 45 relatedness, enhancing traditional hospital infection control strategies. 46 47 Keywords: Healthcare infections, platelet transfusion, septic transfusion, metagenomic 48 sequencing, mNGS 49 50 Abstract word count: 239 51 Text word count: 3132 52 53 3 Abstract 54 Background. Transfusion-related sepsis remains an important hospital infection control 55 challenge. Investigating septic transfusion events is often restricted by the limitations of bacterial 56 culture in terms of time requirements and low yield in the setting of prior antibiotic 57 administration.58 Methods. In three Gram-negative septic transfusion cases, we performed mNGS of direct 59 clinical blood specimens in addition to standard culture-based approaches utilized for infection 60 control investigations. Pathogen detection leveraged IDSeq, a new open-access microbial 61 bioinformatics portal. Phylogenetic analysis was performed to assess microbial genetic 62 relatedness and understand transmission events. 63Results. mNGS of direct clinical blood specimens afforded precision detection of pathogens 64 responsible for each case of transfusion-related sepsis, and enabled discovery of a novel 65 Acinetobacter species in a platelet product that had become contaminated despite 66 photochemical pathogen reduction. In each case, longitudinal assessment of pathogen burden 67 elucidated the temporal sequence of events associated with each transfusion-transmitted 68 infection. We found that informative data could be obtained from culture-independent mNGS of 69 residual platelet products and leftover blood specimens that were either unsuitable or 70 unavailable for culture, or that failed to grow due to prior antibiotic administration. We 71 additionally developed methods to enhance accuracy for detecting transfusion-associated 72 pathogens sharing taxonomic similarity to contaminants commonly found in mNGS library 73 preparations. 74Conclusions. Culture-independent mNGS of blood products afforded rapid and precise 75 assessment of pathogen identity, abundance and genetic relatedness. Together, these 76 challenging cases demonstrated the potential for metagenomics to advance existing methods 77 for investigating transfusion-transmitted infections. 7879 Isolate-b (Platelet bag) n/a n/a 5800012 -K. pneumoniae

Arthropod-borne viruses (arboviruses) pose a significant threat to both human and animal health worldwide. These viruses are transmitted through the bites of mosquitoes, ticks, sandflies, or biting midges to humans or animals. In humans, arbovirus infection often results in mild flu-like symptoms, but severe disease and death also occur. There are few vaccines available, so control efforts focus on the mosquito population and virus transmission control. One area of research that may enable the development of new strategies to control arbovirus transmission is the field of vector immunology. Arthropod vectors, such as mosquitoes, have coevolved with arboviruses, resulting in a balance of virus replication and vector immune responses. If this balance were disrupted, virus transmission would likely be reduced, either through reduced replication, or even through enhanced replication, resulting in mosquito mortality. The first step in mounting any immune response is to recognize the presence of an invading pathogen. Recent research advances have been made to tease apart the mechanisms of arbovirus detection by mosquitoes. Here, we summarize what is known about arbovirus recognition by the mosquito immune system, try to generate a comprehensive picture, and highlight where there are still gaps in our current understanding.

Culex quinquefasciatus mosquitoes are a globally widespread vector of multiple human and animal pathogens, including West Nile virus, Saint Louis encephalitis virus, and lymphatic filariasis. Since the introduction of West Nile virus to the United States in 1999, a cumulative 52,532 cases have been reported to the CDC, including 25,849 (49.2%) neuroinvasive cases and 2456 (5%) deaths. Viral infections elicit immune responses in their mosquito vectors, including the RNA interference (RNAi) pathway considered to be the cornerstone antiviral response in insects. To investigate mosquito host genes involved in pathogen interactions, CRISPR/Cas9-mediated gene-editing can be used for functional studies of mosquito-derived cell lines. Yet, the tools available for the study of Cx. quinquefasciatus-derived (Hsu) cell lines remain largely underdeveloped compared to other mosquito species. In this study, we constructed and characterized a Culex-optimized CRISPR/Cas9 plasmid for use in Hsu cell cultures. By comparing it to the original Drosophila melanogaster CRISPR/Cas9 plasmid, we showed that the Culex-optimized plasmid demonstrated highly efficient editing of the genomic loci of the RNAi proteins Dicer-2 and PIWI4 in Hsu cells. These new tools support our ability to investigate gene targets involved in mosquito antiviral response, and thus the future development of gene-based vector control strategies.